Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Remarks:
The study was requested by ECHA; ECHA decision number: CCH-D-2114359364-45-01/F.
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The aim of this OECD TG 414 study was to assess possible adverse effects on pregnant females and embryo-foetal development which could arise from repeated exposure of Direct Blue 264 via oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised 25 female Wistar rats in the control (C), the low dose (LD), the medium dose (MD) and the high dose (HD) groups.
The following doses were evaluated: 0 (control), 250, 500 or 1000 mg/kg body weight/day.

GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Test System
Species/strain: Healthy Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age of the females
at the arrival at
BSL Munich: approx. 11-12 weeks old
Age of the males at
the start of pairing: between 12 weeks and not older than 15 weeks
Body weight
before initiation
of pairing: males: 302 – 373 g
(mean: 338.87 g, ± 20 % = 271.09 – 406.64 g)
females: 177 – 247 g
(mean: 210.27 g, ± 20 % = 168.22 – 252.32 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to the German Act on Animal Welfare the animals were bred for experimental purposes.
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22  3 °C
- Relative humidity: 55  10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet (contains no phytoestrogens) for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in type III H, polysulphone cages on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). During the pre-mating period and after mating, males were housed in groups (up to 5 animals / cage) in type IV cages.
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least 5 days)

Number and Sex of the Animals
156 animals (52 males and 104 females) were included in the study.


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 2 % Chremophor in aqua ad injectionem
Details on exposure:
Characterisation of the Vehicle
The vehicle used in this study was selected based on the test item’s characteristics. The vehicle to be used in this study was 2 % Chremophor in aqua ad injectionem (sterile water).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (Eurofins Munich Study No. 180640).
Study pre start stability analysis was included on the samples from high dose and low dose group and the investigation was made for 10 days at 20 ± 5 °C (RT – room temperature), 10 days at 5 ± 3 °C (2-8 °C) and 10 days at -20 ± 5 °C.
Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
As the test item was shown to be homogenous at the tested concentrations, but considered to be a suspension according to Eurofins Study No. 180640, samples in this study were taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of prepared formulations of the control group in the first and last weeks of the study (20 samples).
Concentration Analysis
Concentration analysis of formulation samples was determined at three concentrations, 25 mg/mL, 50 mg/mL and 100 mg/mL in study weeks 1 and in the last week of the study. The mean recoveries observed for the LD dose group was between 82.7 % and 117.8 % of the nominal value, between 90.9% and 117.2 % for the MD dose group and between 102.3 % and 118.6 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 100.3 %, 104.1 %, and 110.4 % of the nominal concentration, respectively.
Nominal concentrations were confirmed for all dose groups, as measured mean concentrations were within acceptance criterion of 15 %. All runs were valid in analytical point of view, as run QC samples were within acceptance criteria.
Homogeneity
Homogeneity of formulation samples was determined at three concentrations, 25 mg/mL, 50 mg/mL and 100 mg/mL, in study weeks 1 and the last week of the study.
The coefficients of variation of the different sampling locations (top, middle, bottom) was between 0.1 % and 5.4 % in LD dose group, between 1.2 % and 5.4 % in MD dose group and between 1.7 % and 3.4 % in HD dose group. All samples were homogenous, as COV was below or equal 15 %.
Details on mating procedure:
Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the female was checked to confirm the pregnancy. The day on which sperms were observed in the vaginal smear was considered as gestation day ‘0’. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights were comparable with each other. Each animal was assigned a unique identification number. After getting 100 sperm positive females, the remaining females and males were discarded without any observations.
Duration of treatment / exposure:
Direct Blue 264 at dose levels of 250, 500, and 1000 mg/kg body weight per day was administered on gestation days 5 to 19.
Frequency of treatment:
Once daily.
Duration of test:
On gestation day (GD) 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Group 1 (control).
Dose / conc.:
250 mg/kg bw/day
Remarks:
Group 2.
Dose / conc.:
500 mg/kg bw/day
Remarks:
Group 3.
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Group 4.
No. of animals per sex per dose:
25 females/dose.
Control animals:
yes, concurrent vehicle
Details on study design:
Justification for the Selection of the Test System
This test is performed on the rat. Although several mammalian species may be used, the rat is the preferred rodent species.
This study provides information concerning the effects of prenatal exposure on the pregnant test animal and on the developing organism; this may include death, structural abnormalities, or altered growth and an assessment of maternal effects.
The test item is orally administered daily in graduated doses to several groups of pregnant females from gestation day (GD) 5 to gestation day (GD) 19. During the administration period the animals are observed each day for signs of toxicity. Animals that die or are euthanised for animal welfare reasons during the test are necropsied as well as all surviving animals which are necropsied at the end of the study.
On GD 20, i.e. the day prior to the expected day of delivery, the presumed pregnant females are subjected to a caesarean section, the uterus is removed carefully and weighed. Foetuses are removed from the gravid uterus, identified, sexed, weighed and examined for any external, visceral and skeletal abnormality. The number of corpora lutea, implantations and resorptions are also counted.
The study was requested by ECHA; ECHA decision number: CCH-D-2114359364-45-01/F.

Justification for the Selection of the Test Method
No valid and accepted in vitro method is available for assessing prenatal developmental toxicity potential of the test items.

Examinations

Maternal examinations:
Preparation of the Animals
After the acclimatisation period of at least 5 days, females were paired with males as per the ratio of 1:2 (male to female). Prior to the start of the mating a detailed clinical observation outside the home cage was made. Only healthy animals were used for the study.

Body Weight and Food Consumption
All animals were weighed once before initiation of pairing to ensure that the body weights are within + 20 % variation.
The sperm positive females were weighed during gestations days 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.
Food consumption of sperm positive females was measured on gestations days 5, 8, 11, 14, 17 and 20.
Food consumption was not measured for males during the entire study or for both male and females during the mating period.

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.




Ovaries and uterine content:
Post-Mortem Examination
On gestation day 20, sperm positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using anesthesia (ketamine/xylazine).
None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.
At the time of termination, the dam (presumably pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.

Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appear non-gravid were further examined by staining with 10 % ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with the cervix was weighed.

Thyroid/parathyroid glands from all dams were preserved in 4 % neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation.
The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths as well as the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was also recorded.
Males were sacrificed without any observations at any time after the completion of the mating or were used for other studies.

Evaluation of Thyroid Hormones
Thyroid hormones levels from all dams were assessed at the end of the treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site and was stored under appropriate conditions. Blood samples were assessed for serum levels for thyroid hormones (T3, T4, and TSH).

Histopathology
A full histopathology was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups which were sacrificed at the end of the treatment period.


Fetal examinations:
Foetal Evaluations
All foetuses from a particular dam were identified by using numbered plates and were weighed and sexed based on the anogenital distance. Each foetus was examined for external anomalies and the anogenital distance (AGD) of each foetus was measured. Foetal body weight measured on gestation day 20 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / Cube root of foetus weight).
Particular attention was paid to the reproductive tract which was examined for signs of altered development. External foetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male foetuses.

One half of each litter was examined for soft tissue anomalies by a microdissection technique.
The remaining foetuses were processed by Alizarin staining and the first 20 litters per group were examined for skeletal alterations.
Craniofacial examination of the heads of the foetuses used for the soft tissue examination of the first 20 litters per group were performed for internal structure including the eyes, brain, nasal passage and tongue by razor blade serial sectioning technique.

External Examination
Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.

Skeletal Examination
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95% ethanol. These foetuses were processed using the Alizarin red staining technique. After fixation in 95 % ethanol, the foetuses were macerated with a 1% aqueous potassium hydroxide solution for 1 day and transferred to an Alizarin red solution (0.0025 % in 1 % aqueous potassium hydroxide) for 1 day. After that the foetuses were again transferred to 1 % KOH. Alizarin stained foetuses were then cleared and dehydrated in a solution containing 2 parts of 70 % ethanol, 2 parts of glycerin and one part of benzyl alcohol for 1 day and finally preserved in a 1:1 solution of 70 % ethanol and glycerin.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centers. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centers and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

Visceral Examination
Foetuses scheduled for the visceral examination were pinned to a paraffin covered petri dish with the ventral side up under a stereo microscope. The abdominal and thoracic cavities of all foetuses were dissected and examined for visceral anomalies.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The kidneys were further observed for the presence of clear fluid-filled cysts, cortical cysts, pitting or granular appearance and then sectioned with a sharp scalpel blade to examine the pelvis for distention or the presence of calculi or white granular material. The left kidney was sectioned with one longitudinal slice just off center and the right kidney was sectioned with one transverse slice directly through the papilla. The capsule, cortex, medulla, renal papilla, and renal pelvis were checked for the presence and the pelvis for distension with fluid.
The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.

The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart was recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus. For an examination of the internal anatomy of the heart, the heart was then repositioned and two cuts through the right ventricle were made using micro-dissecting scissors.
The first cut was taken starting from the right of the ventral midline surface at the apex to the base of the pulmonary artery and the second cut was made through the midline surface at the apex extending to the left ventricle in to the ascending aorta. Incisions were opened with fine forceps for the examination of interventricular and auriculo ventricular septum.
After the completion of the visceral examination by the microdissection technique, foetuses were transferred to plastic bottles containing formalin-aceto-alcohol for later craniofacial examination by the razor-blade-serial-section technique.

Craniofacial Examination
Before initiating the serial sectioning with a razor blade, foetuses were transferred to the beaker containing tap water for deformalisation. After deformalisation, a single foetus was decapitated and the head of the fetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.


Statistics:
Evaluation of Results and Statistical Analysis
The findings of this study were evaluated in terms of the observed effects.
Maternal and foetal test results, including an evaluation of the relationship between the exposure of the animals to the test substance and the incidence and severity of all findings as well as findings of foetal external, soft tissue and skeletal anomalies are reported.
Body Weight and Food consumption were presented for intervals during gestation days 0-5, 5-8, 8-11, 11-14, 14-17 and 17-20.
The number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, live and dead foetuses and resorptions, AGD, litter size and litter weights were summarized in tabular form.
The relative weights of thyroid/parathyroid glands were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.
All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).
Indices:
The number of live births and post implantation loss, the number of pups with grossly visible abnormalities, the number of implantations, corpora lutea, live and dead foetuses and resorptions, AGD, litter size and litter weights were summarized in tabular form.
The relative weights of thyroid/parathyroid glands were calculated in relation to the body weight (measured at necropsy) and were presented as percentage.
Historical control data:
Historical Control Data are available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicological relevance observed in the females of any treatment group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality was observed during the treatment period and all animals survived until end of the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weight remained unaffected by treatment with the test item and increased with the progress of the study in the control, LD, MD and HD groups throughout the study period. No statistical significance was achieved in any treatment groups on any day or interval of body weight measurement and all values in the treatment groups were comparable to the controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Although there was significant effect observed in treatment groups on few occasions, it could be attributed to initial low individual food consumption values from few animals and group mean values before initiation of the treatment during gestation day 0-5. In the light of no or marginal effect in HD group (13 % and 0.5% lower) on food consumption during the advanced gestation period (GD 14-17 and 17-20) and no effect on body weight development, this statistically significant effect on food consumption during gestation days was not considered to be biologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
In all terminally sacrificed females, no statistically significant or toxicologically relevant effect was observed on group mean T3, T4 and TSH hormone levels and values were comparable with the controls.
Statistical analysis of post fixed thyroid/parathyroid weights from all dams revealed no statistically significant differences in the absolute and relative (to body weight) thyroid/parathyroid weights of the dose groups when compared to the control group.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Early or late resorptions:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Dead fetuses:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted for any prenatal parameters including terminal body weight, adjusted maternal weight (carcass weight), uterus weight, net weight change from gestation day 0, number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Remarks on result:
other: 1000 mg/kg bw/day was the highest dose tested

Maternal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no test item-related effects of toxicological relevance observed for the mean foetus weight, male and female foetus weight on litter basis (group mean of individual litter mean) in any of the treatment groups when compared with the controls. However, marginal but statistically significantly lower mean male and female foetus weight was observed on individual basis (Sum of weight of all foetuses in group divided by total number of foetuses in respective group) in MD and HD group when compared with the controls. As this difference was marginal (3.8 % lower in MD and 2.9 % lower in HD), not dose dependent and in the light of no significant effect on male and female foetus weight observed based on the litter means, it was not considered biologically relevant.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No test item-related effects of toxicological relevance were noted number of corpora lutea, implantation sites, early and late resorptions, number of live foetuses, number of male and female foetuses, sex ratio (% males), and percent pre- and post-implantation loss in treatment groups when compared to the controls. No dead foetuses were noted in any of the groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
There were no external abnormalities considered to be of toxicological relevance in any of the dose groups. Statistical analysis of data revealed no significant differences compared to the control group.
Low incidences of haematoma on head (1 in in control), misshapen head (1 in control) and umbilical hernia (1 in HD) were noted in isolated foetuses of the control group and/or the dose groups without dose dependency. As these findings were observed mostly in single foetuses, they were considered to be incidental in nature and unrelated to the treatment.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Skeletal examination of the Alizarin red stained foetuses revealed a range of findings which occurred at an incidence generally comparable to or slightly lower or higher in the dose groups when compared to the control group.
Statistical analysis of skeletal data revealed no statistical significance for litter incidences in dose groups when compared with the control.

Slightly higher litter incidences, but without achieving statistical significance for incomplete ossification of skull - basioccipital (80 % compared to 70 % in controls and 5 % in HCD), skull- hyoid body, (20 % compared to 10 % in controls and 20 % in HCD), skull – interparietal (90 % compared to 60 % in controls and 90.91% in HCD), increased ossification of orbital socket region of skull (65 % compared to 50 % in controls and 5 % in HCD ), caudal shift of pelvic girdle (B) (25 % compared to 20 % in controls and 35 % in HCD), caudal shift of pelvic girdle (L) (20 % compared to 15 % in controls and 22.22 % in HCD), misaligned ossification of 4th sternebra (10 % compared to 0 % in controls and 13.33 % in HCD), dumbbell ossification of thoracic vertebral centrum (25 % compared to 15 % in controls and 26.09 % in HCD), full 14th rib (B) (25 % compared to 5 % in controls and 35 % in HCD), rudimentary 14th rib (L) (70 % compared to 50 % in controls and 89.47 % in HCD) and rudimentary 7th cervical rib (L) (20 % compared to 10 % in controls and 30 % in HCD) were observed in the HD group when compared to the control group.

The observed incomplete ossification without achieving statistical significance of a few bones and a few other skeletal findings in the HD group were either marginally higher or within historical control data range. Generally delayed ossification might be correlated to the lower pup weight observed in the HD group, and is not regarded to persist postnatally and not associated with long-term consequences on survival, general growth and development and therefore is not considered to be adverse.
Rudimentary/short ribs are considered as transient abnormalities. In contrast, full/long ribs are considered permanent and may cause health effects in humans. However, postnatal consequences in rats are unknown but are assumed to be minimal. Furthermore, full, rudimentary/short ribs finding values were within the historical control data range and therefore not considered to be treatment-related but spontaneous in nature.

The finding of a caudal shift of the pelvic girdle (bilateral) in HD group was well within historical control data range (35 %) and changes of the position of the pelvic girdle relative to the number of pre-pelvic vertebrae can occasionally be seen in animals of this strain and therefore this finding was not considered to be adverse.

There was no statistical significance and no indication of a test item-related trend in the type and/or litter incidences of other skeletal findings and they were therefore considered to be spontaneous in nature. Frequencies for litter incidences of few skeletal findings were even less in the treated groups compared to the controls. Therefore, these findings are not considered to be treatment-related but rather solely spontaneous in nature.
Visceral malformations:
no effects observed
Description (incidence and severity):
Internal observation of the foetal viscera by free hand microdissection technique revealed a range of visceral findings in all groups including control.
Visceral findings observed in the dose groups were at frequencies generally comparable to or in some cases slightly higher or lower in frequency compared to controls. As the observed findings were either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

All litter incidences from dose groups were statistically insignificant when compared with the control. There was a higher litter incidence of umbilical artery transposed, dilatation of ureter, dilatation of renal pelvis and convoluted ureter was observed in all treatment groups including control group. However, values were well within historical control data (HCD) range (85.71 % - umbilical artery transposed, 87.50 % - dilated ureter, 47.37 %- dilated renal pelvis and 73.91 % - convoluted ureter). There was discoloration of a few organs including liver, adrenal, spleen, thymus observed without dose dependency. Discoloration of organs was considered likely to reflect the consequence of a functional disorder and thus not strictly as developmental anomalies.

Due to lack of dose dependency and consistency, these discolouration findings were not considered as toxicologically relevant.
Dilated/convoluted ureter and dilated renal pelvis are common findings in rodent studies and are classified as a variation as they are transient and likely to be postnatally reversible. Furthermore, values were within the historical control data so that no toxicological relevance was attributed to it.
There was also a higher litter incidence of enlarged spleen (20 % in HD and 15 % in control) and long thymus (24% in HD and 10% in control) was observed in HD group compared to the control group. However values were within the historical control data rage.
Other effects:
no effects observed
Description (incidence and severity):
Craniofacial examination by razor blade serial sectioning technique revealed a few predominant findings (ear subcutaneous and subdural haematoma, subcutaneous edema of head, retinal fold and dilated lateral ventricle) at low frequencies generally comparable to or in some cases slightly higher or lower in frequency in the dose groups compared to the controls. but well within the historical control data range. These findings were considered to be spontaneous in nature and not related to the treatment with the test item. Statistical analysis of the data revealed no statistical significance for litter incidence of any of these findings.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 1000 mg/kg bw/day was the highest dose tested..

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
On the basis of this prenatal developmental toxicity study in Wistar pregnant female rats with Direct Blue 264 at dose levels of 250, 500, and 1000 mg/kg body weight per day administered on gestation days 5 to 19, the following conclusions can be made:
No mortality was observed during the treatment period of this study.
All clinical signs observed in terminally sacrificed females were incidental or non- adverse in nature.
No treatment-related effect on body weight development, food consumption, prenatal data parameters, litter data parameters, thyroid weight and hormones, foetal anogenital distance and gross pathology of terminally sacrificed females was observed up to highest dose tested.
Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the HD group.
No effects of Direct Blue 264 on pregnant females and foetuses were found at dose levels up to 1000 mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for both maternal toxicity and foetal toxicity of Direct Blue 264 in this study is considered to be 1000 mg/kg body weight/day (the highest dose tested).
Executive summary:

In this OECD TG 414 study possible adverse effects on pregnant females and embryo-foetal development were assessed, which could arise from repeated exposure of Direct Blue 264 via oral administration (gavage) to female rats during gestation days 5 to 19. Nulliparous and non-pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on the day of sperm positive vaginal smears (GD 0). The 4 groups comprised 25 female Wistar rats in the control (C), the low dose (LD), the medium dose (MD) and the high dose (HD) groups.

The following doses were evaluated: 0 (control), 250, 500 or 1000 mg/kg body weight/day.

No mortality was observed during the treatment period of this study.

All clinical signs observed in terminally sacrificed females were incidental or non- adverse in nature.

No treatment-related effect on body weight development, food consumption, prenatal data parameters, litter data parameters, thyroid weight and hormones, foetal anogenital distance and gross pathology of terminally sacrificed females was observed up to highest dose tested.

Furthermore, no treatment-related and toxicologically relevant external, visceral or craniofacial findings were observed in the HD group (1000 mg/kg bw/day).

No effects of Direct Blue 264 on pregnant females and foetuses were found at dose levels up to 1000 mg/kg body weight/day. The NOAEL (No Observed Adverse Effect Level) for both maternal toxicity and foetal toxicity of Direct Blue 264 in this study is considered to be 1000 mg/kg body weight/day (the highest dose tested).